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Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus
Ana Claudia A. Melo
1,a, Denise Valle
c, Ednildo A. Machado
d, Ana Paula Salerno
a,
Gabriela O. Paiva-Silva
a, Narcisa L. Cunha E Silva
b, Wanderley de Souza
b,
Hatisaburo Masuda
a,*aDepartamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21944-590,
Brazil
bLaborato´rio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Rio de Janeiro 21944-590, Brazil
cDepartamento de Entomologia, Fundac¸a˜o Instituto Oswaldo Cruz, Instituto de Biologia do Exercito, Rio de Janeiro, Brazil dLaborato´rio de Entomologia Me´dica, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro
21944-590, Brazil
Received 11 August 1999; received in revised form 24 January 2000; accepted 25 January 2000
Abstract
The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [35S]-methionine or 32Pi and the secretion of newly synthesized ovarian
vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.2000 Elsevier Science Ltd. All rights reserved.
Keywords: Ovary; Synthesis of vitellogenin; Follicle cells; Rhodnius
1. Introduction
Since embryonic development of insects occurs in iso-lation from the maternal body, the egg can survive only if it contains all necessary material for the embryo’s
Abbreviations: O-Vg=ovarian vitellogenin; VT=vitellin; Vg= vitellog-enin; DEAE=diethylaminoethyl; LBTI=lima bean trypsin inhibitor;
32Pi=[32P]-Pi (radioactive inorganic phosphate); SBTi=soybean trypsin
inhibitor; SDS=sodium dodecyl sulphate; SDS-PAGE=Polyacrylamide gel electrophoresis in the presence of SDS; DME=Dulbecco’s modified Eagles’s medium; NP40=(octylphenol)-polyethoxyethanol; NBT= Ni-troblue tetrazolium; BCIP=bromo-chloro-indoyl phosphate; PBS=phosphate-buffered saline.
* Corresponding author. Fax:+55-21-270-8647.
E-mail address: [email protected] (H. Masuda). 1 Present address: Departamento de Patologia, Centro de Cieˆncias
Patolo´gicas, Universidade Federal do Para´.
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 2 3 - 0
growth. This material, the yolk, composed of proteins, sugars and lipids, is stored in an organized manner inside the egg. In most insects, a single phospholipoglycoprot-ein, vitellin (Vt), is the main component of eggs and serves both for embryonic and in some cases, early larval development (Postlethwait and Giorgi, 1985; Zhu et al., 1986; Oliveira et al. 1986, 1989). Vitellin is derived from vitellogenin (Vg), which in most insects is synthe-sized exclusively by fat bodies (Valle et al., 1993). Oocytes are specialized for accumulating this Vt precur-sors by receptor-mediated endocytosis (as reviewed by Telfer et al., 1982; Raikhel and Dhadialla, 1992).
(Lauverjat et al., 1984) and mosquitoes (Raikhel and Lea, 1991). These channels, which are transient, undergo dramatic changes in patency in Rhodnius during oocyte maturation and this change correlates perfectly with the rate of vitellogenin uptake (Oliveira et al., 1986).
In addition to accumulating Vg synthesized by the fat body the ovaries also synthesize proteins that are required for embryonic development. The commitment of follicle cells to this process is well known (Melius and Telfer, 1969; Anderson and Telfer 1969, 1970). In some organisms the ovary has been described as the site of synthesis of non-vitellin yolk proteins such as paravit-ellogenin in Hyalophora cecropia (Telfer et al., 1981) and egg-specific protein in Bombyx mori (Irie and Yama-shita, 1983). Synthesis of vitellogenin by ovaries so far has been demonstrated only in higher Diptera and some Coleoptera (Postlethwait et al., 1980; Bownes, 1982; Brennan et al., 1982; Fourney et al., 1982; Zhai et al., 1984; Bianchi et al., 1985; Peferoen and De Loof, 1986; Zongza and Dimitriadis, 1988). To our knowledge there are no reports bearing on the possibility of Vg synthesis by ovarian tissues of Hemiptera.
In this work, the ovaries of the hemipteran Rhodnius prolixus are shown to synthesize Vg. Follicle cells sur-rounding large oocytes are active in this process, and it is shown that follicle cells continue to produce Vt even after closure of interfollicular channels, i.e. that are no longer engaged in the uptake of yolk precursors from the hemolymph. A possible role of R. prolixus ovarian Vg is considered.
2. Materials and methods
2.1. Animals
Insects were taken from a colony of Rhodnius prolixus maintained at 28°C and 70–80% relative humidity. The experimental insects were adult mated females fed on rabbit blood at three-week intervals.
2.2. Tissue preparations
Ovaries were dissected out from females fed two days earlier with rabbit blood and the follicles were examined under a Zeiss stereomicroscope. Oocyte lengths were measured using an ocular micrometer. To obtain follicle cells free of oocytes, each follicle was opened up, using iridectomy scissors, and the oocyte cytoplasm discarded, so that the final preparation was a “lawn” of follicle cells attached to a naked oocyte membrane.
2.3. 32Pi purification
Carrier-free 32
Pi purchased from Comissa˜o Nacional de Energia Nuclear (Sa˜o Paulo, Brasil) was purified by
means of Dowex 1X-10 ion-exchange column (de Meis and Masuda, 1974).
2.4. Incorporation of [35S]-methionine or 32Pi into
proteins
Isolated ovaries or follicle cells were incubated in Dulbecco’s Modified Eagle’s medium (DME-M3916, Sigma, St Louis) containing 0.1–0.2µCi/µl [35
S]-meth-ionine (catalog no. 51006 ICN Pharmaceuticals, Inc, Irv-ine, California) or purified32Pi (0.15µCi/µl) in a
phos-phate-free culture medium (DME-D3656, Sigma, St Louis) at 28°C for 90 min. Subsequently, the tissues were set aside for determination of total protein accord-ing to Lowry et al. (1951) and protease inhibitors (1 mM benzamidine; 0.05 mg/ml leupeptin; 0.05 mg/ml SBTI; 0.05 mg/ml LBTI) were added to the medium. The radioactive proteins secreted to the culture medium were analyzed by SDS-PAGE followed by autoradiography or immunoprecipitated with antiserum raised against Vt. In some experiments the proteins secreted to the medium were precipitated with 15% TCA (w/v) washed several times and the radioactivity estimated by scintillation counting. The data were expressed as [35
S] incorporated per microgram of tissue protein.
2.5. Polyacrylamide gel electrophoresis
Electrophoresis was performed in the presence of SDS (Laemmli, 1970) in a 7.5% polyacrylamide gel or using a polyacrylamide gradient (6% to 22%), followed by staining with Coomassie Brilliant Blue. The gels were destained using a mixture of 7% acetic acid and 40% methanol. Molecular masses were estimated using the following protein standards for SDS-PAGE (Sigma, St
Louis): myosin (205 kDa), β-galactosidase (116 kDa),
phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa) and α -lactal-bumin (14 kDa).
2.6. Vitellin purification
and dialyzed overnight against 10 mM Tris–HCl pH 7.4 containing 0.15 M NaCl. The proteins were concentrated on speed-vac concentrator and applied onto a column
(90×1.5 cm) of Sephacryl S-200 HR. The peak of Vt
was collected and re-applied onto the same column but now eluted in a 10 mM Tris–HCl pH 7.4 containing 0.3 M NaCl. The purity of the sample was monitored by SDS-PAGE and only the subunits of Vt were visible.
2.7. Antiserum
Purified vitellin (1 mg) suspended in complete Freund’s adjuvant was injected subcutaneously in the back of a 1.5 kg rabbit. Two weeks after injection, a booster was given; 30 days later blood was taken from an ear vein and the serum examined by Western blotting (Towbin et al., 1979) using an adult female’s hemo-lymph.
2.8. Immunoprecipitation
For immunoprecipitation, samples (100 µl) were pre-incubated with pre-immune serum (20µl) during 60 min at 4°C. Then, 20µl of protein A-agarose (P7786, Sigma, St Louis) was added and allowed to react for 60 min at
4°C. The samples were centrifuged at 12,000 g for 5
min. To the supernatant 20 µl of specific serum raised against Vt was added, followed by incubation during 180 min at 4°C. After addition of 30µl of protein A-agarose, the samples were centrifuged at 12,000 g for 5 min. The immunoprecipitate was washed three times with 50 mM Tris pH 8.0, 0.5 M NaCl, 1% NP40 (octylphenol-polyethoxyethanol) and counted.
2.9. Immunoblotting
The proteins were separated by SDS-PAGE (7.5% polyacrylamide), during 180 min at 2 mA/cm and then electrotransferred to a nitrocellulose membrane in 25 mM Tris, 192 mM glycine, 20% methanol (pH 8.3) for 120 min at 150 mA, followed by staining with Ponceau Red or preparation for immunostaining as follows: the membrane was incubated with antiserum raised against purified vitellin followed by secondary antibody conju-gated with alkaline phosphatase and developed with NBTI/BCIP (Towbin et al., 1979). After immunostaining the membrane was washed several times with water, dried at room temperature and autoradiographed using XAR5 film (Sigma, St Louis).
2.10. Trichloroacetic acid protein precipitation
The radioactive samples (culture medium containing radioactive proteins secreted by follicle cells) were placed on filter paper and dried at room temperature. The filter papers were washed once with cold 15%
trichlo-roacetic acid for 15 min, twice with cold 10% tricholo-roacetic acid and finally with 70% ethanol (Sahal and Yamaguchi-Fujita, 1987). The papers were dried and the radioactivity estimated by scintillation counting.
2.11. Analysis of radioactive vitellogenin by ion-exchange chromatography
Culture medium obtained from follicle cells incubated with [35
S]-methionine or32
Pi was dialyzed during 8 h at
4°C against buffer A (20 mM Tris–HCl pH 8.4, 2 mM
EDTA, 2 mM EGTA, 5 mM NaN3) to remove [35
S]-methionine or 32Pi not associated with proteins. The
dialysate was then applied onto a DEAE-Toyopearl 650
M column (1.4×24 cm) equilibrated in buffer A. The
column was washed with buffer A, a linear gradient from 0.1 M to 0.2 M NaCl in buffer A was developed and the fractions collected. Each fraction was examined in a spectrophotometer at 280 nm and an aliquot counted in a scintillation counter.
2.12. Immunofluorescence
Ovarioles were isolated and fixed using 4% paraform-aldehyde in PBS. The fixed preparations were allowed to adhere to cover glasses coated with poly-l-lysine, then
washed with PBS and treated with 150 mM NH4Cl
dur-ing 20 min. Permeation was obtained by treatment with 0.1% Triton X-100 in PBS, for 5 min at room tempera-ture. Non-specific staining was avoided by treatment with PBS containing 1.5% albumin and 0.5% gelatin (blocking buffer) during 30 min. After incubation with antiserum raised against vitellin (diluted 1:500) for 60 min, preparations were washed with blocking buffer and finally incubated with goat rabbit secondary anti-body associated with fluorescein (Gibco, Grand Island, N.Y) diluted 1:100 in blocking buffer, for 60 min in the dark. The preparation was mounted with 0.2 M n-propyl gallate in 9:1 glycerol-PBS and analyzed using a Zeiss laser scanning microscope (LSM 310), operating in non-confocal mode. Obtained images were all equally pro-cessed using Adobe Photoshop.
2.13. Immuno-electron microscopy localization
For electron microscopy analysis vitellogenic follicles were fixed in a mixture of 0.1% glutaraldehyde type I and 4% paraformaldehyde in PBS (pH 7.2) for 2 h at room temperature. After fixation, oocytes were washed in PBS and then incubated with 50 mM glycine in PBS for 60 min, washed in PBS and dehydrated in a series of methanol solutions (30% to 90%) and finally
embed-ded in Unicryl (British Biocell) at 220°C under UV
illumination. Ultrathin sections were collected on 300 mesh nickel grids. The sections were subsequently
30 min, PBS containing 1.5% albumin, 0.5% of gelatin and 0.1% Tween 20 (blocking buffer) for 30 min, and primary antibody raised against vitellin for 60 min (diluted 1:500). Afterwards, sections were washed in blocking buffers, incubated with 10 nm gold-labeled goat anti-rabbit IgG (1:100) (Sigma Chemical Co) for 60 min, and thoroughly washed in PBS. Grids were examined in a Zeiss 900 electron microscope, after stain-ing with uranyl acetate and lead citrate. Control experi-ments demonstrating specificity were performed using non-immune serum followed by incubation with gold-labeled goat-anti-rabbit IgG.
3. Results
3.1. Follicle cells as a site of synthesis of vitellogenin
When Rhodnius prolixus ovaries, with their associated follicles, were incubated in a culture medium containing [35S]-methionine at 28°C, radioactive vitellogenin and
other proteins were synthesized and secreted to the medium (Fig. 1). In order to localize the site of vitellog-enin synthesis in the ovary, follicular epithelium cells free of oocytes were incubated in the same culture medium. Analysis of the secreted proteins by SDS-PAGE and Western blots shows that the main protein secreted to the culture medium by the whole ovary and by the follicle cells is radioactive (Fig. 1C) and corre-sponds to vitellogenin, since it is recognized by anti-serum raised against vitellin (Fig. 1A and B). This experiment demonstrates that follicle cells are a site for synthesis of this yolk protein.
Rhodnius vitellogenin and vitellin are known to be
Fig. 1. Analysis of vitellogenin synthesized by the ovary and follicular epithelium cells of Rhodnius prolixus. (A) SDS-PAGE stained by Coomassie Blue R. (B) Western blot. (C) Autoradiography of the nitrocellulose membrane in B. Follicle cells or ovaries were incubated for 90 min in DME culture medium in the presence of [35S]-methionine. The proteins secreted to in the culture medium were separated by SDS-PAGE (A, 7.5%
acrylamide), transferred to a nitrocellulose membrane, and challenged with antibody raised against vitellin and developed using a secondary antibody conjugated with phosphatase (B), followed by autoradiography (C). Molecular weights of the four vitellin subunits are indicated at the left. Lane 1: non-radioactive extract of chorionated oocyte; lane 2: proteins synthesized and secreted to the culture medium by a follicle-cell preparations isolated from around 20 oocytes; lane 3: proteins synthesized and secreted to the medium by three intact ovaries.
Fig. 2. Purification of ovarian vitellogenin secreted by the follicle cells. The follicle cells were incubated with [35S]-methionine (I) or 32Pi (s) for 90 min and the proteins secreted to the culture medium
separated on a DEAE-Toyopearl column (see Materials and Methods). The protein content was estimated from the absorbance at 280 nm (—). The radioactivity associated with proteins was estimated by scintil-lation counting. (Inset) Autoradiography of SDS-PAGE of [35S]
phosphorylated lipoglycoproteins (Masuda and Oliveira, 1985). To ascertain whether the vitellogenin synthesized by follicle cells is also phosphorylated, parallel experi-ments were conducted with cells incubated in culture medium enriched with32Pi or [35S]-methionine followed
by separation of the secreted proteins on a DEAE-Toy-opearl column (Fig. 2). The experiment shows that a major radioactive peak is labeled with both [32P] and
[35
S]. Analysis by SDS-PAGE of total radioactive pro-teins or immunoprecipitated with antiserum against vitel-lin also shows that the main protein in fact correspond to vitellogenin (inset).
3.2. Localizing the cells responsible for synthesis
In order to determine whether vitellogenin synthesis occurs in specialized cells of the follicular epithelium, antiserum against vitellin was raised in rabbits and used
Fig. 3. Immunofluorescence of a follicle challenged with serum raised against vitellin. The follicles (1.5 mm to 1.7 mm in length) was fixed with 4% paraformaldehyde in PBS, followed by washing with 150 mM NH4Cl and then 1.5% albumin plus 0.5% of gelatin (w/v). The preparation was
challenged with serum raised against vitellin and then with goat anti-rabbit secondary antibody associated with fluorescein and visualized in a confocal laser scanning microscope (conventional fluorescence mode). Follicle cells and oocyte are indicated on the figure. (A) Phase-contrast micrograph; (B) Control of immunofluorescence of similar follicle treated with secondary antibody; (C) immunofluorescence of follicle treated with antiserum raised against vitellin; (D) Superimposition of images A and C in a RGB system. Red channel corresponds to immunofluorescence (presence of Vg and Vt). Green channel corresponds to phase-contrast micrograph.
for immunolocalization. Goat anti-rabbit antibody lab-eled with fluorescein (Fig. 3) or gold particles (Fig. 4) were used. Independent of the technique used, vitellog-enin was found inside the great majority of follicle cells analyzed and also, as expected, inside the oocyte. Except for the follicle cells found close to the area between two oocytes, where a fluorescent signal of different intensity was observed, no other specialization could be detected among the other follicle cells (Fig. 3, Fig. 4). Controls, using secondary antibody, were performed for both tech-niques. No reactions was observed either by immuno-fluorescence (Fig. 3) or immunogold detection (data not shown).
Fig. 4. Immunocytochemical localization by transmission electron microscopy of vitellogenin in sectioned follicles embedded in Unicryl. Anti-vitellin immune serum was used as primary antibody, followed by incubation with 10 nm gold-labeled goat anti-rabbit IgG. Arrow indicate representative gold particles. The follicle with associated oocyte (1.5 to 1.7 mm in length) was selected from ovary dissected two days after a blood meal. (A) A view inside the follicle cell (FC) (×60,000). (B) Detail of O-Vg secretion between two follicle cells (×112,500). (*) indicates intercellular space. (C) Panoramic view of the region between a follicle cell (FC) and oocyte (O) (×21,000). (D) A view inside the oocyte showing yolk granule (Y) and cytoplasm (cy) (×55,000). (FC) Follicle cell; (MV) Microvilli; (O) Vitellogenic oocyte.
(Fig. 5). The figure shows that the epithelial cells of small follicles are much more active with respect to total protein synthesis than cells of follicles of larger size. In order to determine when the synthesis of vitellogenin occurs, the proteins secreted by epithelial cells derived from follicles of different sizes were analyzed by SDS-PAGE, and autoradiographed (Fig. 6A). The experiment shows that vitellogenin synthesis occurs primarily in the larger follicles, suggesting its participation in the late phase of vitellogenesis. Only the subunit of high molecu-lar mass is visible, probably due to the small amount of methionine in the smaller subunits (see Fig. 1B and C). Additionally the fact that only the largest subunit is vis-ible in Fig. 6 can also be attributed to the quenching of radioactivity. The autoradiography was obtained expos-ing the film on to dried gel for 30 days while in Fig. 1 where the proteins were previously transferred to
nitro-celulose 11 days were enough to sensitize the film and display the small subunits (Fig. 1C). The immunofluo-rescence experiment shown in Fig. (6B and C) where epithelial cells derived from large follicles reacted strongly when challenged with antibody raised against Vt, but not the epithelial cells derived from small size oocytes is in agreement with the biochemical obser-vation (Fig. 6A).
4. Discussion
Fig. 5. Synthesis of proteins by epithelial cells dissected from fol-licles of different sizes. Follicle cells associated with oocytes of differ-ent sizes, as shown on the abscissa, were dissected and washed to remove the egg cytoplasm (see Methods) and then incubated in DME medium containing 0.16µCi/µl [35S]-methionine at 28°C. After
incu-bation for 90 min the culture medium was collected, the proteins were precipitated with 15% trichloroacetic acid and the radioactivity was estimated in a scintillation counter. The data are presented normalized by tissue protein. Vertical bars represent standard errors of five deter-minations.
Fig. 6. Analysis of proteins newly synthesized by epithelium cells obtained from follicles of different sizes. (A) The epithelium cells were incubated for 90 min in DME culture medium containing 0.1–0.2 µCi/µl [35S]-methionine at 28°C. After incubation, the culture medium was
collected and immunoprecipitated with antiserum raised against vitellin. The precipitates were electrophoresed and autoradiographed. The arrow indicates the position of the 180 kDa subunit of vitellogenin. The number above the lanes represent the size in mm of follicles, based on the lengths of the oocytes they originally contained. Before immuno-precipitation, an aliquot was precipitated in acid and counted to adjust the same amount of radioactive proteins in each sample. (B) Phase-contrast micrograph of a field showing large size follicle (L) (1.5 mm) together with small size follicles (S) (,1.0 mm). (C) Immunofluorescence of the same follicles challenged with antiserum raised against Vt.
through receptor-mediated endocytosis (Telfer, 1961; Roth and Porter, 1964; Mundall and Engelmann, 1977; Hagedorn and Kunkel, 1979; Engelmann, 1979; Oliveira et al., 1986). Although Vg in the hemolymph is clearly synthesized by Rhodnius fat bodies (Valle et al., 1993), the present work shows that follicle cells of the ovary are an additional site for Vg synthesis. The Vg synthesis by follicle cells was observed by the incorporation of labeled amino acid or [32
modifi-cations (Kunkel and Nordin, 1985; Raikhel and Bose, 1988; Dhadialla and Raikhel, 1990) or both. The pres-ence of microheterogeneity in the Vt molecule has already been observed (Telfer, 1965; Wyatt and Pan, 1978; Engelmann, 1979; Hagedorn and Kunkel, 1979; Imboden and Law, 1983). It is tempting to speculate that the role attributed to egg specific protein or paravitellog-enin can be accomplished by Rhodnius ovarian Vg. The fact that ovarian Vg is synthesized especially in late stage of vitellogenesis (Fig. 6A–C) suggests that small oocytes might contain no or primarily vitellogenin pro-duced by the fat body while mature oocytes might con-tain both. This possibility is supported by previous work that demonstrated that the uptake of yolk protein paral-lels the degree of patency of the space between follicle cells (Oliveira et al., 1986). This space diminishes in follicles of $1.7 mm and closes completely in follicles of 2.0 mm, but the follicle cells are still very active in the synthesis of vitellogenin (Fig. 6A). This fact suggests that, in large follicles, the uptake of vitellogenin synthe-sized by the fat body is reduced, so the Vg accumulated during the final phase of vitellogenesis is primarily ovarian vitellogenin. Fig. 1 shows that intact ovaries incubated in culture medium also released O-Vg, although the newly synthesized O-Vg was expected to be incorporated by the oocyte. A possible explanation for this is that, under our experimental condition, the rate of O-Vg synthesis and O-Vg uptake is mismatched. We are assuming that, during the incubation of ovaries in vitro, the synthesis of O-Vg by follicle cells are not per-fectly matched with the capacity of oocytes to take up Vg so part of O-Vg can be lost to the incubation medium. This conclusion is based on two facts: the rate of Vg uptake by the oocytes of Rhodnius is in fact much lower in vitro than in vivo (Oliveira et al., 1986) and the synthesis of O-Vg occurs in oocytes of 1.5 mm in length (Fig. 6) before the closure of epithelial channels (Oliveira et al., 1986). Possibly, in vivo, O-Vg is not released to the hemolymph in a great extent since the rate of Vg uptake is much higher.
An interesting question raised by these results is why only cells obtained from large follicles are engaged in vitellogenin synthesis even though the smaller cells have a higher capacity for total protein synthesis (Fig. 5). Clearly the timing for O-Vg synthesis changes in func-tion of the size of the follicle and this quesfunc-tion deserves to be investigated.
Acknowledgements
We wish to express our gratitude to Jose´ de Souza Lima Junior and Jose´ Francisco de Souza Neto for main-taining our colony of Rhodnius prolixus; to Lilian Soares, Heloisa S. Coelho, Rosane O. M. M. da Costa for their technical support in the biochemical work and
to Noeˆmia Rodrigues and Sebastia˜o Cruz for their assist-ance on electron microscopy; and to Dr Martha M. Sor-enson for a critical reading of the manuscript. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Conselho de Aperfeic¸oamento de Ensino Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Programa de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico (PADCT) and Programa de Nu´cleos de Exceleˆncia (PRONEX).
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